Page 92 of 201
preparation for the previous voyage and that were retained in the assembly depot and loaded onto the next available voyage voyage 17. These animals are commonly described
as ‘carried over’ or ‘held over’. A random and representative sample of animals from cohort 8
were sampled on two occasions with the second sampling occurring seven days after the first sampling.
Cohort 12 was a newly arrived group of cattle that were sampled initially on the day of arrival at the assembly depot and then sampled a second time nine days later.
It was not possible to select the same individuals for sampling at the first and second sampling sessions, but animals that were sampled at the second session were selected from
pens containing animals that had been sampled at the first session for each of the two re- sampled cohorts.
Serum samples were collected from animals from 1 voyage voyage 17. Serum and nasal swab samples collected from the same animal in another nested study aimed at comparing
nasal swab test results to serum antibody test results from the same animals. Samples were collected at the time animals arrived at the assembly depot for four separate cohorts of
animals from two states Table 27.
8.3 Detection of respiratory pathogens
One or more of the viruses or bacteria of interest were detected in the nasal swabs from 1,1501,484 77 of cattle. The remaining 3341,484 23 animals were negative for all
organisms tested.
Pathogen prevalence is reported as a percentage of animals tested within each sampled cohort and voyage Table 28 and Table 29. There was significant variation between cohorts
and voyages for prevalence of individual viruses and bacteria p0.05.
Bovine coronavirus BCoV was the most commonly detected virus in nasal swabs from cattle in the assembly feedlot, found in cattle from all voyages and in all but 2 of the cohorts
tested Table 28. Cohort-level prevalence for BCoV varied from 0 to 94 and the overall prevalence when all samples were combined was 40.
Other respiratory viruses were detected at much lower prevalence levels and there were a number of cohorts that had no detections for one or more of the other four viruses Table
28. There were only three occasions where a cohort level prevalence exceeded 10 for the other four viruses: twice for BVDV and once for BRSV.
Page 93 of 201 Table 28: Count of number of nasal swabs by cohort and voyage, number of samples detected positive for each of five respiratory viruses,
prevalence as a percentage and the 95 confidence interval CI for the prevalence.
Cohort Voyage
Total BCoV
BoHV-1 BRSV
BVDV BPIV-3
N n
95 CI n
95 CI n
95 CI n
95 CI n
95 CI
1 10
7 70.0 34.8-93.3
0.0 0.0-30.8 0.0
0.0-30.8 0.0
0.0-30.8 0.0 0.0-30.8
2 200
188 94.0 89.8-96.9
0.0 0.0-1.8
2 1.0
0.1-3.6 9
4.5 2.1-8.4
2 1
0.1-3.6 3
200 117
58.5 51.3-65.4 0.0
0.0-1.8 0.0
0.0-1.8 12
6.0 3.1-10.2
1 0.5
0.0-2.8 Voy 5
410 312
76.1 71.7-80.1 0.0
0.0-1.0 2
0.5 0.06-1.8
21 5.1
3.2-7.7 3
0.7 0.2-2.1
4 102
58 56.9 46.7-66.6
0.0 0.0-3.6
0.0 0.0-3.6
1 1.0
0.0-5.3 0.0
0.0-3.6 5
112 2
1.8 0.2-6.3
0.0 0.0-3.2
0.0 0.0-3.2
0.0 0.0-3.2
0.0 0.0-3.2
6 99
56 56.6 46.2-66.5
0.0 0.0-3.7
0.0 0.0-3.7
1 1.0
0.0-5.4 1
1 0.0-5.5
7 100
6 6.0
2.2-12.6 4
4.0 1.1-9.9
1 1.0
0.03-5.4 0.0
0.0-3.6 1
1 0.0-5.4
Voy 8 413
122 29.5 24.2-34.2
4 1.0
0.3-2.5 1
0.2 0.0-1.3
2 0.5
0.1-1.7 2
0.5 0.1-1.7
8a 25
2 8.0
1.0-26.0 2
8.0 1.0-2.6
0.0 0.0-13.7
3 12.0 2.5-31.2
0.0-13.7 8b
18 0.0
0.0-18.5 1
5.6 0.1-27.3 0.0
0.0-18.5 1
5.6 0.1-27.3
0.0 0.0-18.5 9
92 41
44.6 34.2-55.3 5
5.4 1.8-12.2 0.0
0.0-3.9 4
4.3 1.2-10.8
4 4.3 1.2-10.8
10 20
0.0 0.0-16.8
0.0 0.0-16.8 0.0
0.0-16.8 2
10.0 1.2-31.7 0.0 0.0-16.8
11 75
7 9.3
3.8-18.3 2
2.7 0.3-9.3
0.0 0.0-4.8
2 2.7
3.2-9.3 2
2.7 0.3-9.3
12a 143
32 22.4 15.8-30.1
0.0 0.0-2.5
0.0 0.0-2.5
7 4.9
2.0-9.8 4
2.8 0.8-7.0
12b 108
28 25.9 18.0-35.2
1 0.9 0.02-5.1
4 3.7
1.0-9.2 1
0.9 0.0-5.1
2 1.9
0.2-6.5 13
80 4
5.0 1.4-12.3
0.0 0.0-4.5
0.0 0.0-4.5
1 1.3
0.0-6.8 4
5 1.4-12.3
Voy 17 561
114 20.3 17.1-23.9
11 2.0
1.0-3.5 4
0.7 0.2-1.8
21 3.7
2.3-5.7 16
2.9 1.6-4.6
14 100
47 47.0 36.9-57.2
0.0 0.0-3.6
10 10.0 4.9-17.6
0.0 0.0-3.6
0.0 0.0-3.6
Total 1,484
595 40.1 37.6-42.6
15 1.0
0.6-1.7 17
1.1 0.7-1.8
44 3.0
2.2-4.0 21
1.4 0.9-2.2
Page 94 of 201 Table 29: Count of number of nasal swabs by cohort and voyage, number of samples detected positive for each of four bacterial respiratory
pathogens, prevalence as a percentage and the 95 confidence interval CI for the prevalence.
Cohort Voyage
Histophilus somni Mycoplasma bovis
Mannheimia haemolytica Pasteurella multiocida
N n
95 CI n
95 CI n
95 CI n
95 CI
1 10
1 1.0
0.3-44.5 0.0
0.0-30.8 0.0
0.0-30.8 6
60.0 26.2-87.8
2 200
14 7.0
3.9-11.5 0.0
0.0-1.8 88
44.0 37.0-51.1
121 60.5
53.4-67.3 3
200 53
26.5 20.5-33.2
0.0 0.0-1.8
9 4.5
2.1-8.4 44
22.0 16.5-28.3
Voy 5 410
68 16.6
13.1-20.5 0.0
0.0-1.0 97
26.7 19.6-28.1
171 41.7
36.9-46.6 4
102 5
4.9 1.6-11.1
0.0 0.0-3.6
5 4.9
1.6-11.1 20
19.6 12.4-28.6
5 112
11 9.8
5.1-16.9 0.0
0.0-3.2 6
5.4 2.0-11.3
50 44.6
35.2-54.3 6
99 49
49.5 39.3-59.7
5 5.1
1.7-11.4 36
36.4 26.9-46.6
33 33.3
24.2-43.5 7
100 58
58.0 47.7-67.8
0.0 0.0-3.6
7 7.0
2.9-13.9 15
15.0 8.6-23.5
Voy 8 413
123 29.8
25.4-34.4 5
1.2 0.4-2.8
54 13.1
10.0-16.7 118
28.6 24.3-33.2
8a 25
23 92.0
74.0-99.0 6
24.0 9.4-45.1
0.0 0.0-13.7
7 28.0
12.1-49.4 8b
18 13
72.2 46.5-90.3
3 1.7
3.6-41.4 1
5.6 0.1-27.3
5 27.8
9.7-53.5 9
92 59
64.1 53.5-73.9
25 27.1
18.4-37.4 6
6.5 2.4-13.7
43 46.7
36.3-57.4 10
20 2
10.0 1.2-31.7
0.0 0.0-16.8
1 5.0
0.1-24.9 2
6.7 1.2-31.7
11 75
53 70.7
59.0-80.6 0.0
0.0-4.8 0.0
0.0-4.8 1
1.3 0.0-7.2
12a 143
99 69.2
61.0-76.7 11
7.7 3.9-13.3
8 5.6
2.4-10.7 19
13.3 8.2-20.0
12b 108
78 72.2
62.8-80.4 19
17.6 10.9-26.1
26 24.1
16.4-33.3 10
9.3 4.5-16.4
13 80
17 21.3
12.9-31.8 3
3.8 0.8-10.6
0.0 0.0-4.5
7 8.8
3.6-17.2 Voy 17
561 344
61.3 57.1-65.4
67 11.9
9.4-14.9 42
7.5 5.4-10.0
94 16.8
13.8-20.1 14
100 87
87.00 78.8-92.9
0.00 0.0-3.6
6 6.00
2.2-12.6 4
2.00 1.1-9.9
Total 1,484
622 41.9
39.4-44.5 72
4.8 3.8-6.1
199 13.4
11.7-15.2 387
26.1 23.9-28.4
Page 95 of 201
BCoV was present in 92 of single viral detections and 92 of mixed viral-bacterial detections.
The individual animal prevalence of BCoV in our study was similar to that recently reported for feedlot cattle on the south coast of New South Wales with clinical BRD. Other studies
have reported similar prevalence measures in clinically normal feedlot cattle.
33
There is conflicting evidence of association between presence of coronavirus and respiratory disease in cattle. Some studies suggest an increased risk of respiratory disease in cattle that
are shedding BoCV in nasal secretions.
34
Our findings from necropsies conducted on voyages suggested that the presence of BoCV in nasal secretions was linked to an increased odds of death due to respiratory disease during
the voyage.
35
Other studies have not found an association.
36
Serological testing for BCoV antibodies was not performed as part of the study reported here due to a lack of a commercially available test kit. Other studies suggest that animals that are
shedding BoCV in nasal secretions may be likely to have low serologic titres against BoCV. Animals with low antibody titres and shedding virus may be more susceptible to infection
with these viruses and therefore the animals in our study that were shedding BoCV may be considered at risk of developing some form of respiratory infection with this pathogen.
Bovine viral diarrhoea virus BVDV was detected in nasal swabs from 44 out of 1,484 3 animals, with the highest prevalence estimates occurring in a cohort of Western Australian
carry-over cattle 12 and south Australian slaughter cattle 10. BVDV was detected in nasal swabs and lung samples from animals that died during voyages but was not
significantly associated with fatal respiratory disease.
37
The seroprevalence of BVDV in this study 56; Table 38, was consistent with previous reports for Australian cattle with seroprevalence estimates ranging from 45 to 77
38
, and also with the “approximately 60” reported for Western Australian live export cattle in 1985.
39
Seroconversion during the feeding period has been linked to an increased likelihood of requiring treatment for BRD.
40
Thirteen out of 334 animals were nasal swab positive and seronegative for BVDV, and 3 out of 334 animals were nasal swab positive and seropositive. These animals were either
transiently infected and captured immediately prior to, or soon after, mounting an immune response, or they were persistently infected PI. Persistently infected animals have viral
antigen in their nasal secretions and are mostly seronegative.
33
Hasoksuz et al. 2002; Fulton et al. 2011
34
Lathrop et al. 2000; Hasoksuz et al. 2002; Thomas et al. 2006; Fulton et al. 2011
35
Moore et al. 2014
36
Cho et al. 2001; Hasoksuz et al. 2005
37
Moore et al. 2014
38
Dunn et al. 1995; Taylor et al. 2006
39
Littlejohns and Horner 1990
40
OConnor et al. 2001
Page 96 of 201
In feedlots PI animals can shed BVDV and infect susceptible contact animals pen mates. Acute BVDV infection is well characterised in cattle and is known to impair immune cell
function and predispose animals to secondary bacterial infections of the respiratory tract. PI animals therefore represent a threat to other healthy animals in the immediate area
particularly if they have no immunity against BVDV.
Consideration should therefore be directed at detection and management of potential PI animals during the assembly period to minimise the risk of BVDV infection and subsequent
development of BRD. It is also important to note that the application of a single PACE test to remove PI animals does not guarantee the removal of BVDV circulation from a live export
consignment.
41
Bovine herpesvirus 1 prevalence was similar to that observed in cattle at feedlot entry in the U.S.
42
Bovine herpesvirus 1 is known to play an important role in the development of BRD through both direct tissue damage and immunosuppressive effects that allow secondary bacterial
infections to cause respiratory disease. However, nasal shedding of BoHV-1 is not necessarily associated with respiratory disease or reduced performance in feedlot cattle.
43
Results from voyage necropsies conducted as part of this project did not find an association between BoHV-1 in nasal or lung samples and mortality due to BRD.
44
Thirty-nine percent 122309 of newly received animals were seropositive for antibodies to BoHV-1. This is within the range of previously reported seroprevalences for Australian beef
cattle 13-85.
45
The 61 of animals that were seronegative in our sampling are likely to be susceptible to infection by pen-mates shedding BoHV-1. No significant increase in BoHV-1 nasal
prevalence was recorded for newly received animals sampled at feedlot entry and again 9 days later.
These findings suggested that BoHV-1 did not play a primary role in the development of BRD during voyages in the cattle we studied.
Bovine respiratory syncytial virus BRSV is recognised as a common primary pathogen in respiratory disease in young calves
46
and adult dairy cattle.
47
BRSV has not previously been detected in nasal swabs from beef feedlot cattle over the age of 4 months.
48
In this study the significant increase in BRSV nasal prevalence between depot entry and re-resting 9 days later cohorts 12a and 12b is likely to indicate
transmission of BRSV to naïve animals during this period.
41
Moore et al. 2014
42
Fulton et al. 2002; Storz et al. 2000
43
Fulton et al. 2002
44
Moore et al. 2014
45
Dunn et al. 1995; Dunn et al. 2000
46
Sacco et al. 2013
47
Bidokhti et al. 2012
48
Moore 2014
Page 97 of 201
The seroprevalence of BRSV in the study reported here was 46. This is higher than the 27 prevalence reported in a serosurvey of Australian feedlot cattle
49
, and lower than the 100 reported in an outbreak of respiratory disease in New South Wales.
50
The seroprevalence for BPIV-3 87 was the highest out of the four viruses. This, combined with a low nasal prevalence 1.4 and lack of evidence for an association
between BPIV-3 and respiratory disease in Australian live export cattle
51
, or Australian feedlot cattle
52
, suggests that BPIV-3 plays a secondary role, if any, in the development of BRD in Australian live export cattle.
Two of the respiratory bacteria were detected from every cohort and voyage with an overall combined prevalence of 42 for Histophilus somni and 26 for Pasteurella multocida while
cohort prevalence estimates ranged from 1-92 H. somni and from 1-61 P. multocida Table 29.
The overall prevalence for detection of Mannheimia haemolytica was 13, there were four cohorts that had no M. haemolytica detected and the cohort level prevalence in those
cohorts that did detect M. haemolytica ranged from 4.5-44.
There was relatively little Mycoplasma bovis detected. The overall prevalence was 5, 9 of 16 cohort-samples were completely negative and the prevalence in the seven cohort
samples where M. bovis was detected ranged from 2-27.
Previous studies examining the presence of bacteria in nasal swab samples have used bacterial culture for isolation and identification of bacteria. Our approach involved the use of
PCR detection of bacterial DNA and did not involve bacterial culture. Bacterial culture is considered to be less sensitive than PCR when detecting presence of bacteria in swab
samples.
53
PCR may also return a positive result even when there are no live or viable bacteria present because it is capable of detecting non-viable fragments of bacterial
genome. Understanding some of the different characteristics of different methods is important when comparing our findings to those from other papers that may have used
different techniques.
53
Histophilus somni can be carried in the upper respiratory tract in normal, healthy animals without disease. H. somni is also a potential cause of a number of disease syndromes
including fibrinopurulent pneumonia, myocarditis and polyarthritis-serositis.
54
The individual animal and cohort level prevalence estimates for H. somni in the current project were both approximately 42. This is higher than previous reports where individual
animal prevalence ranged from 0-9.
55
Previous studies have not found a direct association between nasal isolation of H. somni at feedlot entry and risk of subsequent respiratory disease in feedlot cattle.
56 49
Dunn et al. 1995
50
Hick et al. 2012
51
Moore et al. 2014
52
Dunn et al. 1995; Dunn et al. 2000
53
Fulton and Confer 2012
54
Griffin et al. 2010
55
Allen et al. 1991; Corbeil et al. 1986; Fulton et al. 2002; Van Donkersgoed et al. 1994
56
Allen et al. 1991; Fulton et al. 2002
Page 98 of 201
Results from voyage necropsies conducted during this project found relatively high prevalence of H. somni detection in live export cattle that died during voyages but no
association between detection of H. somni and either histological evidence of pneumonia or death due to BRD.
57
The prevalence of Mannheimia haemolytica was 13 at the individual level and 10 at the cohort level. These findings were consistent with previously reported prevalence estimates
for M. haemolytica in nasal swabs from clinically normal feedlot cattle.
As for other bacteria, M. haemolytica is found as a commensal organism of the nasopharynx and tonsils of healthy cattle, even though it is capable of causing respiratory disease under
favourable conditions.
Pasteurella multocida was detected in nasal swabs from 26 of cattle with a cohort level prevalence of 27. These findings were consistent with previously reported prevalence
estimates for P. multocida in nasal swabs from clinically normal feedlot cattle.
58
As for other bacteria under investigation in this project, P. multocida is found as a commensal organism of the nasopharynx and tonsils of healthy cattle, even though it is
capable of causing respiratory disease under favourable conditions.
58
The presence of P. multocida in nasal swabs therefore does not necessarily mean increased risk of respiratory
disease in those animals. One study on lung tissue or swabs from beef cattle at necropsy collected between 1994 and
2002 detected a trend over that time period towards an increased isolation of P. multocida and reduced isolation of M. haemolytica as the principle bacterial pathogen associated with
BRD.
59
Our findings from necropsy samples collected form cattle that died during export voyages found that P. multocida was isolated more frequently than M. haemolytica in lung and nasal
swab samples.
60
There are a number of possible explanations for this trend including changes in bacterial virulence and antimicrobial resistance, changes in the efficacy of available vaccines and
antibiotics, reduced age of cattle at feedlot entry, changes in the way sick cattle are identified and treated, and increased use of mass medication programs.
61
Mycoplasma bovis M. bovis is an important cause of pneumonia, arthritis and tenosynovitis in feedlot cattle.
62
M. bovis was detected in nasal swabs from 5 of cattle in our study. Previously papers have reported prevalence estimates of M. bovis in apparently healthy feedlot calves range from 0-
43.
63
57
Moore et al. 2014
58
Fulton et al. 2002; Allen et al. 1991
59
Welsh et al. 2004
60
Moore et al. 2014
61
Rice et al. 2007; Welsh et al. 2004
62
Caswell et al. 2010
63
Allen et al. 1991; Hanzlicek et al. 2011; White et al. 2010; Wiggins et al. 2007
Page 99 of 201
The role of M. bovis in the development of BRD remains relatively undefined in the scientific literature. In our study, the findings from necropsy samples collected during the voyage
suggested that M. bovis was significantly associated with mortality due to respiratory disease during voyages.
64
Multiple organisms were detected in nasal swab samples from many animals in this study. Overall, a single bacteria only was detected in 5031150 43.7 animals, while one or more
viruses and bacteria were detected in 434 37.7 animals, and one or more viruses in 213 18.5 animals.
Spearman correlation coefficients were used to look for statistical associations between presence of multiple pathogens in the same swab sample.
The presence of BCoV was significantly correlated with the presence of BRSV, M. haemolytica, P. multocida and H. somni p0.01. Bovine viral diarrhoea virus was present in
8 of mixed infections but was not significantly correlated with any other viruses or bacteria p0.05. The presence of BRSV was significantly correlated with the presence of H. somni
p0.001. Bovine herpesvirus 1, BVDV and BPIV-3 were not significantly correlated with any other organism.
Histophilus somni was present in 77 of single bacterial detections and 54 of mixed viral- bacterial detections. The presence of H. somni was significantly correlated with the presence
of BCoV, BRSV, M. bovis p 0.001 and P. multocida p 0.05. P. multocida was present in 33 of single bacterial detections and 51 of mixed viral-bacterial detections. The presence
of P. multocida was significantly correlated with the presence of BCoV p0.001, M. haemolytica p0.001 and H. somni p0.05.
It is not surprising that co-infections were common given our understanding of the way that respiratory disease develops in cattle. Co-shedding of BCoV and BRSV has not been
reported previously, to the best of our knowledge.
Two cohorts were each sampled twice to look for change in viral and bacterial detection over time Cohort 8 and Cohort 12.
Cohort 8 represented animals that had been carried over in the assembly feedlot from the previous voyage preparation. The second sampling showed a reduction in prevalence or no
change for all five viruses, but the change was not statistically significant for any of these comparisons.
Cohort 12 was sampled for the first time at the point of arrival at the assembly feedlot and then again several days later. There was no evidence of any significant change for four of
the five viruses. The exception was for BRSV, where the second prevalence 3.7 was significantly higher than the first prevalence 0.0, p=0.03.
For bacterial pathogens there was no difference between repeat samples in Cohort 8 p0.05.
64
Moore et al. 2014
Page 100 of 201
In Cohort 12, there was a significant increase in the prevalence of M. bovis p=0.02 and M. haemolytica p0.001 over time while there was no difference in prevalence of H. somni
p0.05 or P. multocida p0.05.
The increase in prevalence in repeated samples from the same group over time is likely to be due to the proliferation of commensal bacteria following inhibition of the immune system
secondary to environmental stressors, for example transportation, co-mingling, and inter- animal transmission of viruses and bacteria. It is also possible that the results may simply
reflect variation over time without any real underlying change in proliferation or carriageinfection rates.
Our results showed a rise in prevalence when the same group of animals was sampled at assembly depot entry and resampled nine days later rise from 8 to 18, suggesting that
there was proliferation and or spread of bacteria within and between animals.
8.4 Screening of explanatory factors against nasal prevalence
